Potential Use of Biomphalaria alexandrina Snail Antigens for Serodiagnosis of Schistosomiasis Mansoni by Immunoblot Analysis

Background The aim of this study was to evaluate the possible use of Biomphalaria alexandrina snail antigens in diagnosis of schistosomiasis mansoni using enzyme linked immunolectrotransfere blot (EITB). Methods S. mansoni adult worm crude antigens (AWA), feet and visceral humps of B. alexandrina and Bulinus truncatus were used. Hyperimmune mice sera (HIS) versus each antigen were prepared for diagnosis of S. mansoni using western blot (WB). Results Snail foot antigens were more specific in antibodies detection than visceral hump antigens. Three of five polypeptides of B. alexandrina foot antigen identified by S. mansoni HIS showed specific positive reactivity. These polypeptides were at MW of 31/32 and 43 kDa. While, only one of the six polypeptides of B. alexandrina hepatopancrease antigen identified by S. mansoni HIS, at a MW of 43 kDa was specific. Similarly, 2 polypeptides at MW of 44 and 55 kDa were specific in detection of anti- S. haematobium antibodies. However, the antigenically active polypeptide of B. truncatus hepatopancrease antigen had no specific reactivity towards anti-S. haematobium antibodies. Conclusion B. alexandrina foot antigens were the most specific of the tested snail antigens in diagnosis of schistosomiasis mansoni.

Introduction espite widespread efforts for schistosomiasis management, it remains a main health dilemma in tropical and subtropical districts (1). Serodiagnosis is essential for diagnosis of parasitic diseases with its precision being affected by types and extent of purification of the used antigens (2). Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) as a method of purification, overcome the cross-reactivity between diverse antigenic components and augmented the antigenic yields by increasing the number of antigens which might be present in little amounts (3). Newly emerging antigenic fractions strongly linked to human species were found specific diagnostic tools. This is evident in schistosomiasis where antigenic sharing between adults, immature stages and intermediate hosts was established (4). Establishment of trematodes infection in their definitive hosts is confirmed by demonstration of certain circulating antibodies detected against antigens of their snail intermediate hosts (5). Sera of patients infected with S. mansoni demonstrated antibodies against hepatopancrease of B. glabrata snails infected with homologous cercaria. The researchers explained that the glycocalyx of Schistosoma cercariae contains antigens contracted from B. glabrata snail tissues (6). Sulahian et al. (7) considered that using EITB is an accurate mean of detection of specific Schistosoma antibodies. The current study was directed to detect the value of B. alexandrina snail antigens in diagnosis of schistosomiasis mansoni using EITB.

Preparation of snail antigens
B. alexandrina and B. truncatus medium to large sized snails were collected from Abu-Rawash, Giza and reared in the laboratory for production of laboratory-bred snails according to EL-Bahy (8). . After 4-6 weeks snails were dissected into two parts, a foot and visceral hump. These were separated, homogenized, sonicated and left overnight for extraction (9).The protein content of the prepared antigens was measured (10), divided into aliquots and stored at -20 °C until used.

Preparation of Schistosome adult worm crude antigen
Cercariae of S. mansoni and S. haematobium (Egyptian strain) were provided by Schistosome Biological Supply Program Unit (SBSP), Theodor Bilharz Research Institute, Giza, Egypt. They were obtained from experimentally infected B. alexandrina and B. truncatus snails. Twenty to thirty cercariae were injected intra-peritoneal in each Swiss albino mice (22 mice, 15-20 gm each) and 8 weeks later, adult worms were collected from liver and premesenteric veins (11). They were homogenized, sonicated then centrifuged at 20000 rpm for 1 hour at 4 °C. The supernatant containing the crude antigens was distributed into 1ml aliquots in plastic vials after measuring its protein content as above and stored at -20 °C until used (12).

Preparation of specific hyper-immune mice sera:
Mice HIS were prepared against snail and parasite antigens according to Langly and Hillyer (13) via initial subcutaneous injection of an equal volume of Freund ̓ s complete adjuvant mixed with 0.4 mg protein for each antigen and injected subcutaneously at different places at back of mice. After 3 weeks, another 0.4 mg of protein for each antigen, mixed in an equal volume of Freund ̓ s incomplete adjuvant and divided into 3 doses, injected intramuscularly at biweekly intervals. Ten to fourteen days later, the blood was collected from retro-orbital vein using capillary tubes under ether inhalation anesthesia. Serum separation was obtained by centrifugation at 3000 rpm for 5 minutes. The level of specific antibodies in sera of immu-D nized mice was evaluated. Negative control sera were obtained by bleeding mice prior to immunization.

SDS-PAGE and electrophoretic transfer:
The protein components of S. mansoni adult crude worm antigens and snails' antigens were separately resolved by SDS-PAGE under reducing conditions using 12% SDS-PAGE (100µg/Lane) (Bio Rad System; USA) according to Laemmli (14). After the electrophoresis, the resolved polypeptides were electrophoretically transferred to a nitrocellulose membrane for immunoblotting using a transfer apparatus (15).The antigen-blotted nitrocellulose membrane was cut vertically into strips of 15x0.5 cm. One strip was incubated with one serum sample diluted 1:50 in 2 ml blocking solution (1% skimmed milk in 0.1 M phosphate buffered saline, pH 7.4, containing 0.0.2% Tween 20 (EL Gomhoreya pharmaceutical CO.) for 2 h at room temperature (RT) with gentle rocking, washed five times with blocking solution, and then incubated for 2 h at RT with peroxidase conjugated goat anti-mouse IgG antibody. The conjugate was used at a concentration of 1:500 in blocking buffer (Sigma, Immunochemical). After washing, the strips were incubated in (amino-9-Ethyl carbazole; AEC) for 30 minutes. The strips were then washed with distilled water to stop the reaction, air-dried and examined for color development at the site of positive reacting fractions using software Gel pro-analyzer.

Results
Fractionation of S. mansoni snail and adult worm crude antigen revealed multiple components via SDS-PAGE. Adult S. mansoni crude antigen revealed at least 9 polypeptides. Those polypeptides molecular weights ranged from16-120 kDa. Of them, 7 protein bands corresponding to molecular weights ( (Fig.2,  Lane4). No polypeptides were identified by preimmunized mice sera (Fig.2, Lane 5).Therefore, the polypeptides at MW of 31/32 and 43 kDa were considered to be specific in detection of S. mansoni antibodies.  All of them were recognized by S.mansoni HIS except that at 40 kDa (Fig.3, Lane 3). From these polypeptides, five at MW of 93,80,70,55,52 reacted crossly with S. haematobium HIS (Fig.3, Lane 4). Pre-immunized mice sera recognized polypeptides at MW of 55 and 80 kDa (Fig.3, Lane 5). Thus only one polypeptide at MW of 43 kDa was specific in detection of anti-S. mansoni antibodies. Concerning B. truncatus foot antigen, its homologous HIS recognized bands at 44 and 55 kDa (Fig.4, Lane 2). Both polypeptides were identified by S. haematobium HIS (Fig.4, Lane 3). It did not react crossly with S. mansoni HIS (Fig.4, Lane 4) or with pre-immunized mice sera (Fig.4, Lane 5). Thus, 2 polypeptides at MW of 44 and 55 kDa were specific in detection of anti-S. haematobium antibodies. On the other hand, the antigenically active polypeptide of B. truncatus visceral hump antigen had no specific reactivity towards anti-S. haematobium antibodies (Fig.5, Lane 2). Similar antigenic profiles were observed with both S. haematobium HIS (Fig.5, Lane 3) and S. mansoni HIS (Fig.5, Lane 4). No cross reactivity with pre-immunized mice sera was detected.

Discussion
In tropical and subtropical regions, schistosomiasis attains a chief health concern (16). In Egypt, the Nile valley area is highly endemic for schistosomiasis with infection rates beyond 80% bringing Egypt to be one of the most endemic countries in the world (17). Researches based on developing new antigens for diagnosis of schistosomiasis focused on improving the extent of sensitivity and specificity of the selected antigen which in turn enhanced the accuracy of the serological test used (3). The possibility of the use of shared antigens between trematodes and their intermediate hosts for serodiagnosis is apparent in schistosomiasis depending on the described antigens shared between schistosomes and their intermediate hosts (18). The current study was directed to detect the value of Schistosome snail antigens in diagnosis of schistosomiasis mansoni using EITB. The antigenic community between snails and their trematode parasites was already investigated and it was suggested that molecular mimicry of some of the shared antigens manifests a genetic accommodation between the parasite and its host (19).
In our study a variety of antigens were prepared from S. mansoni adult worms and their snail intermediate host (B. alexandrina foot and visceral hump antigens) and from S. haematobium adult worms and their snail intermediate host (B. truncatus visceral hump and foot antigens). They were used to clarify the antigen antibody relation between schistosomes and their related snails. The current study clarified the antigenic similarity between S. mansoni and its snail intermediate hosts from the aspect of molecular weights using SDS-PAGE and from the aspect of immune reaction using WB technique after preparation of HIS. Our results revealed the presence of 7 major protein bands of the fractioned S. mansoni AWA corresponding to molecular weights of 92, 70, 67,54,44,30 and 20 kDa. These results showed some similarity to Rupple et al. (20) who reported that in all chronically old infected mice, antibodies against S. mansoni AWA proteins of 67 kDa were prominent. When sera of S. mansoni infected patients were analyzed by immunoblotting during the prepatent infection, anti-adult IgG antibodies elucidated an intense reaction recognizing the area of 30-40 kDa (21). In addition, Soliman et al. (22) used SDS-PAGE to analyze soluble worm antigens, cercarial antigen preparations and soluble egg antigens of S. mansoni. The authors reported that 32 kDa was a chief band of these three fractionated antigens. In the present work, we found that fractionation of B. alexandrina foot antigen revealed the presence of 9 major protein bands at MW of 109,102,94,69,54,50,32,28 and 22 kDa. While, that of B. alexandrina visceral hump antigen revealed the presence of 10 major protein bands at 103, 94, 93, 85, 69, 64, 54, 50, 36.5 and 33 kDa. These results showed that the AWA and both B. alexandrina antigens have a common polypeptide band at MW of 54 kDa. When comparing the same snail species, Shalaby et al. (23) reported that B. alexandrina snail foot and hepatopancrease antigens contained 8 and 7 polypeptides by SDS-PAGE respectively.
Their molecular weights ranged from 21-97 kDa. They added that the component of 97 and 30 kDa were major bands in B. alexandrina foot while, that of 66 and 30 kDa were major bands in B. alexandrina hepatopancrease. The potential of B. alexandrina antigens was studied by Gamal-Eddin et al. (24) who concluded that immunization of albino mice with purified F iv 20-29 kDa &F v 20-24 kDa antigenic fractions of B. alexandrina resulted in reduction of the worm burdens, the total eggs in tissues and reduced the histopathological changes in the liver. The MWs of these polypeptides are a little bit different from the ranges of MWs of foot and visceral extracts revealed by our study, which may be attributed to differences in the antigen preparations, techniqual differences, use of different concentrations of the resolving gel and different concentration of proteins by different researchers.
In the present study we evaluated the specificity of Schistosoma snail foot and visceral hump antigens in antibody detection of S. mansoni by evaluating their reaction versus specific prepared HIS at a dilution of 1:50 via WB technique. Our results indicated that B. alexandrina foot antigen was more specific than B. alexandrina hepatopancrease antigen in antibody detection. Where, three of five polypeptides of B. alexandrina foot antigen identified by S. mansoni HIS showed specific positive reactivity. These polypeptides were at MW of 31/32 and 43 kDa. While on the other hand, only one of the six polypeptides of B. alexandrina hepatopancrease antigen identified by S. mansoni HIS, at a MW of 43 kDa was specific. Moreover, several molecules were inhibited as 77/76 and 36/37 kDa components that failed to be recognized by S. mansoni HIS. Also, cross inhibition detected by failed recognition of 77/67, 36/37, 31/32 and 43 kDa molecules by incubation of B. alexandrina foot antigen with S. haematobium HIS, reinforcing the hypothesis of antigenic community between S. mansoni and B. alexandrina foot antigen.
Coinciding with our results, when immunoblotting tests employed to detect AWA antibodies, an immunogenic fraction with a molecular weight of 31-32 kDa (Sm 31/32) was considered to be the most frequently documented fraction. It revealed 98% positivity in the pretreatment stage then declined or became negative in the serum of treated patients exposed to low infection. Thus it is considered of high diagnostic importance and could therefore be used as a serologic marker (25)(26). These studies together with our data specifying 31/32 kDa band may give an evidence that B. alexandrina foot antigen is probably a suitable antigen to replace AWA during immunodiagnosis of schistosomiasis mansoni. We reported no cross reactivity of B. truncatus feet antigen against S. mansoni HIS. In addition, no specific reactivity had been shown by antigenically active polypeptides of B. truncatus visceral hump antigen. The specificity of B. alexandrina foot antigen to their trematodes was studied by Shalaby et al. Our experiment provides a support to the assumption of the antigenic community between S. mansoni and one of its vectors, B. alexandrina, through exhibition of common antigenic epitopes with its intermediate host.
B. alexandrina has the advantage of being an affluent source of antigens for serodiagnostic and prophylactic studies for S. mansoni. It is easy maintained in the laboratory, with a high protein yields and low expenditure, compared to other parasite antigens, as AWA and soluble egg antigens that require completion of the long life cycle. However, the cross-reactivity exhibited between the parasite and the mollusk restricted the specificity of these antigenic epitopes.

Conclusion
Biomphalaria alexandrina foot antigen was the best antigen among the tested ones that can replace S. mansoni adult worm crude antigen thus, proved to have the potential as a screening tool for schistosomiasis immunodiagnosis. However, its sensitivity may necessitate more enhancements by purification of the used antigen. Moreover, the recognition and categorization of these antigenic epitopes is vital for the development of recombinant proteins that could be used in immunodiagnosis of schistosomiasis.Further trials are required in order to better characterize the cross-reactive and non cross-reactive snail antigen epitopes that are preferentially recognized by resistant experimental animals and humans.